A kinase is an enzyme that plays the most important role in regulating in vivo signal transduction systems. Protein kinase enzymes take charge of in vivo signal transduction by transferring a γ-phosphate group of adenosine 5′-triphosphate (ATP) to a substrate protein. Protein kinase enzymes are classified with tyrosine kinases and serine/threonine kinases. More than 500 kinase enzymes have been found so far (G. Manning et al, Science 2002, 298, 1912).
A tyrosine kinase functions to phosphorylate a hydroxyl (—OH) group of a tyrosine residue and take complete charge of functions of in vivo signal transduction systems. When such a kinase enzyme is overexpressed, or overactivated by mutation, various diseases (cancer, diabetes, inflammation, brain disease, etc.) are caused. A living body should harmoniously turn on/off cell signal transduction systems in order to maintain homeostasis. In this case, when signal transduction does not continue to be regulated due to overactivities of certain protein kinase enzymes, diseases such as cancer are caused (P. B. Jensen and T. Hunter, Nature 2001, 411, 355.). Therefore, a molecular probe capable of effectively detecting the activities of certain kinase enzymes is of very high importance since it can be used to diagnose diseases including cancer. Also, such a molecular probe is used to search for a drug having an action mechanism to inhibit the activities of certain overactivated protein kinase enzymes, and thus has a very important situation to develop a new drug.
A method including a series of processes has been known as a method of measuring the presence, overexpression, and activities of such a protein kinase enzyme, but its analytic process is complicated and is also not a method of directly detecting a kinase enzyme but an indirect method of detecting compounds associated with a phosphorylation process, such as ATP. Among these compounds, a fluorescent molecular probe has an advantage in that it can easily detect activities of a kinase enzyme with high sensitivity.
Conventional kinase enzyme fluorescent probes have a very complicated structure. That is, they have a structure in which a fluorophore is introduced into a peptide chain which can be recognized by a kinase enzyme, but have a problem in that it is very difficult to select a suitable fluorophore to cause a change in fluorescence when the enzyme interacts with the peptide chain and to spatially arrange the fluorophore.
Fluorescent probes that can measure the activities of the protein kinase enzymes reported so far have a peptide chain containing a tyrosine residue which can be recognized by the enzyme as described above, and often include a lanthanide metal complex or an organic fluorophore having a complicated structure (Chen. C. et al., J. Am. Chem. Soc. 2001, 124, 3840; Zondlo. S. C. et al., J. Am. Chem. Soc. 2010, 132, 5619; Agnes. R. S. et al., J. Am. Chem. Soc. 2010, 132, 6075; Rhee. H. et al., Angew. Chem. Int. Ed. 2010, 49, 4919; Vaasa. A. et al., Biochemistry and Biophysical Research Communication. 2010, 397, 750; Xu. X. et al., Anal. Chem. 2011, 83, 52; Appelblom. H. et al., Microchim. Acta. 2011, 172, 25; Herbst. K. J. et al., J. Am. Chem. Soc. 2011, 133, 5676; Seong. J. Nat. Comm. 2011, DOI:10.1038/ncomms1414; Lawrence. D. S. Chem Bio Chem. 2007, 8, 373; Tremblay. M. S. et al., Org. Lett. 2008, 10, 5; Sahoo. H. et al., J. Am. Chem. Soc. 2007, 129, 15927; Akiba. H. et al., Chem. Eur. J. 2010, 16, 5018; Shults. M. D. et al., J. Am. Chem. Soc. 2003, 125, 14248; Kikuchi. K. et al., Org. Lett. 2009, 11, 2732; Ojida. A. et al., J. Am. Chem. Soc. 2004, 126, 2454; Wang. Q. et al., J. Am. Chem. Soc. 2005, 127, 7684; Sharma. V. et al., J. Am. Chem. Soc. 2007, 129, 2742).
Also, since the conventional fluorescent probes used to detect a tyrosine kinase includes not only a peptide substrate recognizing the enzyme but a luminophore having a complicated structure to present a fluorescence signal upon recognition of the enzyme, they have a high molecular weight, and a synthesis process is complicated. First of all, when the conventional fluorescent probes detect kinase enzymes in cells or tissues, stability and interference by other compounds may become of a concern, which leads to limitation on their use.
The probes which emit fluorescence when they encounter a certain kinase enzyme as a small molecule still have highly challenging issues in spite of their importance. It is not reported so far that the fluorescent probes can directly detect the presence, overexpression, or activities of the kinase enzyme.
Therefore, the present inventors have developed an epoch-making fluorescent probe, which emits fluorescence through interaction with a tyrosine kinase enzyme in vivo, as a small-molecule probe which is easily synthesized and having high stability and low cytotoxicity in vivo. The fluorescent probe has various advantages (such as high tissue permeability, low fluorescence interference from tissues themselves, high resolution, and low cell injury, etc.) since the fluorescent probe can be two-photon excited. It is confirmed that the developed fluorescent probe has a change in fluorescence when it binds to certain kinase enzymes present in cancer cells, and also exhibits fluorescence when it selectively binds to cancer tissues. As a result, the present inventors have found that the fluorescent probe according to the present invention can be used to image the cells or tissues overexpressing a tyrosine kinase, and image the cancer cells or tissues. Therefore, the present invention is completed based on these facts.